Over the last 15 years, bacterial multi-drug resistance (MDR) has emerged and it has several socio-economical causes, from the use of surface antibacterial agents that are now available in many household products1 to antibiotic over-prescription or failing to complete a course of antibiotics1. Although due to MDR new lines of antibiotics are required, the development of new antibiotics has been reduced by pharmaceutical companies because of the cost and complexity of clinical trials2. Currently, there are relatively few new antimicrobials in development.
The gastrointestinal tract is heavily colonized with an average of 1014 microbes that represent thousands of species, which is 10 times more than the total number of cells in the human body3. More than 90% of this bacterial population falls under two major phyla: Bacteriodetes (a gram-negative phylum) and Firmicutes (a gram-positive phylum)4, 5, while the remaining belong to phyla such as Proteobacteria, and Actinobacteria4, 5. In healthy individuals, microbial diversity in the intestine is stable over time and demonstrates a symbiotic relationship with the host3, but a shift in microbial composition, named dysbiosis, targeting mainly Firmicutes and Bacteroidetes, has been described in several pathologies, including related and non-related gastrointestinal pathologies6-8. For example, microbial dysbiosis in gut is observed in intestinal disorders like intestinal bowel syndrome (IBS), intestinal bowel disease (IBD) and also non intestinal disorders like obesity and type 1 and type-2 diabetes. Specifically, gut microbiota helps to digest food items and various metabolites and chemicals are produced by the resident microbiota, which plays a significant role in host health or disease state. For example, Bacteroides thetaiotaomicron can activate the toll-like receptors (TLRs) in the gut epithelium, which in turn can affect the expression of antimicrobial peptides, such as angiogenins9, 10. In addition to the innate immune system, gut microbiota can also control the host's adaptive immune system through T cell receptor αβ-positive intraepithelial lymphocytes, regulatory T cells and T helper 17 cells5. Overall, gut homeostasis is largely dependent on the normal gut microbiome11.
At the mucosal level the epithelium plays a major role in limiting the passage of bacteria to the sub-mucosa and restricts the presence of bacteria to the gut lumen; cell division is an important factor when the epithelial cells are altered and the epithelium needs to be regenerated12,13. Antimicrobial peptides (AMPs) secreted by epithelial cells have a broad spectrum effect against bacteria and they are part of an ancient defense mechanism that is present in virtually all mammals14. In the gastrointestinal tract, specialized intestinal epithelial cells or circulating inflammatory cells are a major source of these AMPs14. Within the epithelium, Paneth cells are the main producer of AMPs but new data indicate that enterochromaffin (EC) cells can hypothetically also produce certain types of AMPs15.
The EC cells are the major source of chromogranin A (CgA)16, a family of highly acidic proteins. The CgA gene is localized at 14q32 in the human genome, consisting of 8 exons and 7 introns, and its 2-Kb transcript is translated into the 457-residue CgA protein. The overall homology for CgA in different vertebrates is approximately 40%, but the most highly conserved regions occur at the N- and C-termini, which show up to 88% sequence homology. Cell- and tissue-specific CgA processing has been described in the rat, mouse and human GI tract17-19. The CgA primary structure from its cDNA sequence shows the presence of numerous pairs of basic amino acids. These are potential sites for cleavage by prohormone convertases (PC) ⅓ or 2, and carboxypeptidase E/H2, which is consistent with evidence that CgA may serve as a prohormone for shorter bioactive fragments21; this is also suggested by the high sequence conservation of CgA-derived peptides. But in the gut, peptides can be highly sensitive to enzymes present in the luminal environment. Proteolytic fragments of CgA-derived peptides exert a broad spectrum of regulatory activities on the cardiovascular, endocrine and immune systems. Among its highly conserved C-terminal regions, CgA gives rise to a peptide of biological importance: the antihypertensive peptide catestatin (human CTS; CgA352-372)22-24, which has restricted antimicrobial activity against Staphylococcus aureus in vitro25. Similar to other AMPs, CTS can interact with anionic components of fungi and viruses. As a result, the microbial membrane is permeabilized, leading to cell lysis26. In vitro studies have demonstrated that CTS is effective against gram-positive bacteria such as Staphylococcus aureus and group A Streptococcus, gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, yeasts such as Candida albicans and filamentous fungi such as Aspergillus niger, A. fumigates and Trichophyton rubrum26, 27. However, to date, there has been no indication that the in vitro data can be reproduced using an in vivo model, as due to the presence of several enzymes located in the gut lumen, CTS peptide can be rapidly inactivated. Moreover, there is no indication about the type of microblota affected, as the colonic mucosa associated population differs completely for the population present in the feces.
Despite the close association between CTS and Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa in vitro, the effects of in vivo CTS treatment on the different type of gut microbiota are unknown. Our aim was to assess the composition of fecal and colonic mucosa associated microbiota and functional alterations in mice that were exposed to CTS for 6 days.